Project Summary Mitochondrial heterogeneity has been shown to cause two kinds of abnormal cell death responses, called ?incomplete MOMP? (iMOMP) and ?minority MOMP?. MOMP is a critical step in apoptotic cell death that involves the permeabilization of mitochondrial outer membranes. Normally MOMP occurs in most, if not all, mitochondria within the cell, and this results decisively in cell death, as a result of the release of mitochondrial proteins into the cytoplasm leading to the activation of caspases. Caspases are proteases that cleave key protein substrates, which then produce efficient cell destruction. However, under certain situations, this process is inefficient. In the iMOMP scenario, some cells can survive because caspases are inactive and a few mitochondria escape MOMP. iMOMP can contribute to oncogenesis and a failure of apoptosis-based therapy. Minority MOMP is a related scenario, in which caspases can be activated normally, but cells receive a sublethal cell stress that causes MOMP to occur in only a low percentage of the cell's mitochondria, leading to caspase- mediated genome instability and tumorigenesis. Both iMOMP and minority MOMP arise as a result of heterogeneous mitochondrial content of the anti-apoptotic Bcl-2 protein, but the mechanism of heterogeneity is still unknown. The goals of this project are to understand how mitochondrial heterogeneity arises, how it affects iMOMP and minority MOMP, with the eventual goal of understanding how it impacts oncogenesis. To discover proteins controlling mitochondrial heterogeneity, we conducted a focused high-content siRNA screen of ~1300 genes related to mitochondrial function. The hits in this screen included multiple genes connected to mitochondrial quality control (which includes the processes of mitophagy and mitochondrial fission and fusion). Therefore, our central hypothesis is that mitochondrial quality control processes function to limit mitochondrial heterogeneity and thus to limit heterogeneous MOMP responses. We further hypothesize that cancer cells can acquire deficiencies in mitochondrial quality control and that those deficiencies can result in the insidious phenomena of iMOMP and minority MOMP. The proposed studies will determine, for genes involved in mitophagy and other systems involved in mitochondrial quality control, how defects in these genes lead to mitochondrial heterogeneity, and how these genes affect the overall cellular sensitivity to mitochondrial apoptosis. Cancer cells can frequently encounter sublethal concentrations of therapeutic drugs and therefore would be predicted to undergo minority MOMP, with a resulting increase in DNA damage and genome instability. Therefore, it is important to understand how mitochondrial heterogeneity arises and, ultimately, how to reduce it, as an adjunct to existing cancer therapies. 15